Effect Of Canine Interferonγ–inducible GTPases On Cellular Resistance Against Toxoplasma Gondii

Toxoplasma gondii, an obligate intracellular parasite, can cause toxoplasmosis. One third of people was infected with Toxoplasma, while the percentage of Chinese is about 5%-10%. To evade the pressure of host immune system and the fatal effector in the host, T. gondii goes to the place where the immunology system is frail. For example, the invasion of animal or human brain through the blood-brain barrier can cause neurologic symptoms; T. gondii passing across the blood-ocular barrier,may result in visual impairment; T. gondii also pass through the blood-placenta barrier, inducing abortion of humans and animals. There is still no effective vaccine to prevent the occurrence of the disease at present. Therefore, toxoplasmosis is a serious threat to human health and the development of animal husbandry. T. gondii invaded into cells attached to the membrane of host cells, forming parasitophorous vacuole, not combining with host cell endomembrane system, in which the parasite is exempt from the exposure to nature immune factors. In addition, studies have shown that the invasion of pathogen to the host will induce expression of pro-inflammatory cytokine and other innate immune molecules like IFNγ, which can interact with their receptors on the surface of cells, inducing the translation of a large number of endogenous innate immune proteins, which play an important role in resisting the infection of Toxoplasma gondii. Immune effectors like immune related GTPase(IRGs) and guanine nucleotide binding protein(GBPs) are capable of localization to parasitophorous vacuole membrane, which can form a membrane attack complex(MAC)-like structure that is vital for the elimination of the parasite, causing the disruption of the parasitophorous vacuole(PV). This kind of damage to PV may bring about the release of the parasite into cytoplasm and the eradication by the innate immune system.First of all, IRGM4, IRGM5 and IRGM6, fused with GST tag, were expressed to prepare the recombinant proteins as antigens in this study, and ICR mice were immuned by this kind of recombinant proteins, to obtain specific polyclonal antibodies against IRGM4, IRGM5 and IRGM6.Secondly,forward and reverse sgRNAs, designed specifically for the knockout of IRGM4, IRGM5 and IRGM6, were constructed. The cell of MDCK was transfected with pMJ920, expressing cas9 protein, and the plasmid transcribing sgRNA, to obtain the cell lines that the genes of IRGM4,IRGM5 and IRGM6 were knocked out. The experiments of western blot were performed to verify the knockout of corresponding genes.Finally, the comparation of the number of the parasites in the parasitophorous vacuole membrane in IRGM4 ko, IRGM5 ko and IRGM6 ko with normal MDCK with and without the inducement of IFNγwere performed. This study shows that IRGM4, IRGM5 and IRGM6 play an important role in the inhibition of the growth of T. gondii in parasitophorous vacuole membrane.There was no significant difference in the growth of T. gondii between the knockout cell lines of IRGM4, IRGM5 and IRGM6 and normal MDCK cells in the absence of IFNγ. Therefore, the presence of a single IRGM5 and IRGM4 did not entail the MDCK cells to resist the infection of T. gondii, the IRGs family maybeassembly to be a complex to perform the function of resistance to the infection of T. gondii.